CN103361306B - Pluripotent stem cell is separately cultured in mammalian tissues - Google Patents
Pluripotent stem cell is separately cultured in mammalian tissues Download PDFInfo
- Publication number
- CN103361306B CN103361306B CN201310169114.4A CN201310169114A CN103361306B CN 103361306 B CN103361306 B CN 103361306B CN 201310169114 A CN201310169114 A CN 201310169114A CN 103361306 B CN103361306 B CN 103361306B
- Authority
- CN
- China
- Prior art keywords
- culture solution
- cell
- culture
- pluripotent stem
- stem cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The present invention provides pluripotent stem cells in mammalian tissues to be separately cultured, and belongs to technical field of stem cell culture.Under sterile, the separation of tissue mechanical separation method, culture sheep fetal kidney tissue primary cell are cultivated 48 hours with culture solution II 37；It washs after cell clone appearance, is cultivated 18 hours after culture solution III is added；The cell being collected by centrifugation culture solution II suspension cell, is inoculated in culture dish, cell clone occurs；It is frozen after 3~4 generations of biography spare.Culture solution I component includes DMEM, L-Glutamine, nonessential amino acid, Sodium Pyruvate, micro beta -mercaptoethanol, LIF, bFGF；Culture solution II component includes culture solution I, FBS；Culture solution III component includes culture solution I and trypsase.The present invention can from mammalian tissues isolated pluripotent stem cell.
Description
Technical field
The present invention relates to one kind, and pluripotent stem cell method is separately cultured from mammalian tissues organ.
Background technique
Currently, well known had immunomagnetic beads by the method for separating pluripotent stem cell in second trimester amniotic fluid from mammal
Method, two phase cultivations.And both methods cannot be separately cultured pluripotent stem cell from people and mammalian tissues organ.
The present invention provides a kind of pluripotent stem cell isolated culture method, and this method uses low concentration pancreatin culture solution culture mammal
Primary cell is organized, to be separately cultured pluripotent stem cell in tissue.
Summary of the invention
In order to be separately cultured pluripotent stem cell from mammalian tissues, the present invention provides a kind of method, this method energy
It is enough that the stem cell with totipotency potential is separately cultured from mammalian tissues.
The technical solution that the present invention is separately cultured pluripotent stem cell from mammalian tissues is: in the dry thin of serum-free
Low concentration trypsase is added in born of the same parents' culture solution.It is dry to can get pluripotency with this culture solution culture mammalian tissues primary cell
Cell.Wherein, the stem cell medium of serum-free can guarantee to organizer for pluripotent stem cell in cell cultivation process
Do not break up, low concentration trypsase plays a kind of screening effect to pluripotent stem cell in tissue.
The invention has the advantages that pluripotent stem cell can be separately cultured from mammalian tissues.Mammal
Pluripotent stem cell present in tissue is many diseases such as genetic defect, tumour and the exogenous virus for studying mammal
Infect the ideal cell of mechanism.
Detailed description of the invention
The present invention is further described with example with reference to the accompanying drawing
Fig. 1 is sheep fetal kidney tissue pluripotent stem cell；
Fig. 2 is the real-time quantitative amplification curve of sheep fetal kidney tissue pluripotent stem cell expression Oct4, SSEA-1,
In, 2.1 be reference gene glyceraldehyde 3-phosphate dehydro-genase gene (GAPDH), and 2.2 be SSEA-1, and 2.3 be Oct4；
Fig. 3 is the embryoid that sheep fetal kidney tissue pluripotent stem cell In vitro Suspension is formed after culture 7 days.
Specific embodiment
The specific steps that sheep fetal kidney tissue pluripotent stem cell obtains in Fig. 1 are as follows:
1. matching liquid
+ 1% Sodium Pyruvate of+1% nonessential amino acid of culture solution I:DMEM+1%L- glutamine+micro beta -mercaptoethanol
+ 5ng or 500u/ml LIF+5ng/ml bFGF is filtered with the filter of 0.22 μ l, 4 DEG C of preservations；Culture solution II: culture solution I+
15%FBS；Culture solution III: culture solution I+0.01% trypsase is filtered with the filter of 0.22 μ l, 4 DEG C of preservations.
2. sheep fetal kidney tissue pluripotent stem cell is separately cultured
Under aseptic condition, using the separation of tissue mechanical separation method, culture sheep fetal kidney tissue primary cell, culture solution
It is cultivated 48 hours under the conditions of II37 DEG C of 5%CO2 saturated humidity；It is washed 3 times after cell clone appearance with D-PBS, training is then added
Nutrient solution III is cultivated 18 hours under the conditions of 37 DEG C of 5%CO2 saturated humidities；Cell is collected by centrifugation in 1500r/min × 5min, with culture
Liquid II suspension cell is blown, is inoculated in 6cm culture dish with 105 cells/ware, occurs cell clone (Fig. 1) after 72 hours；Pass 3
It is frozen after~4 generations spare.
Fig. 2 the specific steps are
Sheep fetal kidney tissue totalRNA is extracted, genomic DNA removal reaction is carried out before reverse transcription, then reverse transcription is closed
At its cDNA, internal reference is glyceraldehyde 3-phosphate dehydro-genase (GAPDH), primer information be shown in Table 1, Real Time PCR according to
TaKaRa SYBR PremixEX TaqTM IIkit illustrates to carry out, 3 repetitions (Fig. 2) of each sample.
Genomic DNA removal reaction:
Two-step method real time PCR standardization program:
1 Real Time PCR primer sequence of table
Fig. 3 the specific steps are
Take growth conditions good, the good 5th generation sheep fetal kidney tissue pluripotent stem cell of form reaches to cell growth
When 80% convergence degree, under aseptic condition, D-PBS is washed 3 times, and pancreatin digests suspension cell, and 1500r/min × 5min is collected by centrifugation carefully
Cell is resuspended in born of the same parents, embryoid culture solution (DMEM+15%FBS), and adjustment cell concentration is 1 × 106/ml；It is added in 6cm culture dish
15ml D-PBS is a droplet with 20ul and is added dropwise on the inside of ware lid；Ware lid reversing is covered on ware, wet in 37 DEG C of 5%CO2 saturations
Suspend culture under the conditions of degree；After 7d, sterile working collects droplet in 6cm culture dish, until microscopically observation, (figure of taking pictures
3)。
Claims (1)
1. a kind of method that pluripotent stem cell is separately cultured from mammalian tissues, it is characterized in that:
1) match liquid
+ 1% Sodium Pyruvate of+1% nonessential amino acid of culture solution I:DMEM+1%L- glutamine+micro beta -mercaptoethanol+
500u/ml LIF+5ng/ml bFGF is filtered with 0.22 μm of filter, 4 DEG C of preservations；Culture solution II: culture solution I+15%FBS；
Culture solution III: culture solution I+0.01% trypsase is filtered with 0.22 μm of filter, 4 DEG C of preservations；
2) sheep fetal kidney tissue pluripotent stem cell is separately cultured
Under aseptic condition, using the separation of tissue mechanical separation method, culture sheep fetal kidney tissue primary cell, culture solution II
37 DEG C of 5%CO2It is cultivated 48 hours under the conditions of saturated humidity；It is washed 3 times after cell clone appearance with D-PBS, culture is then added
Liquid III, 37 DEG C of 5%CO2It is cultivated 18 hours under the conditions of saturated humidity；Cell is collected by centrifugation in 1500r/min × 5min, with culture
Liquid II suspension cell is blown, with 105Cell/ware is inoculated in 6cm culture dish, cell clone occurs after 72 hours；Passed for 3~4 generations
After freeze it is spare.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310169114.4A CN103361306B (en) | 2013-04-25 | 2013-04-25 | Pluripotent stem cell is separately cultured in mammalian tissues |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310169114.4A CN103361306B (en) | 2013-04-25 | 2013-04-25 | Pluripotent stem cell is separately cultured in mammalian tissues |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103361306A CN103361306A (en) | 2013-10-23 |
| CN103361306B true CN103361306B (en) | 2019-09-06 |
Family
ID=49363568
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310169114.4A Active CN103361306B (en) | 2013-04-25 | 2013-04-25 | Pluripotent stem cell is separately cultured in mammalian tissues |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103361306B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101402943A (en) * | 2008-11-17 | 2009-04-08 | 中山大学中山眼科中心 | Method for in vitro abduction and cultivation of multi-potentiality stem cell |
| CN101864451A (en) * | 2009-04-15 | 2010-10-20 | 中国科学院上海生命科学研究院 | Hoofed mammal inducible multipotential stem cell and preparation method thereof |
| CN101984050A (en) * | 2009-09-01 | 2011-03-09 | 中国科学院广州生物医药与健康研究院 | Cell type used for producing induced pluripotent stem (iPS) cells and preparation method and application thereof |
-
2013
- 2013-04-25 CN CN201310169114.4A patent/CN103361306B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101402943A (en) * | 2008-11-17 | 2009-04-08 | 中山大学中山眼科中心 | Method for in vitro abduction and cultivation of multi-potentiality stem cell |
| CN101864451A (en) * | 2009-04-15 | 2010-10-20 | 中国科学院上海生命科学研究院 | Hoofed mammal inducible multipotential stem cell and preparation method thereof |
| CN101984050A (en) * | 2009-09-01 | 2011-03-09 | 中国科学院广州生物医药与健康研究院 | Cell type used for producing induced pluripotent stem (iPS) cells and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 诱导的多潜能干细胞类胚体分化过程中残留未分化细胞的研究;王书军，等;《中国美容医学》;20111130(第11期);1730-1734 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103361306A (en) | 2013-10-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Abecasis et al. | Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors: bioprocess intensification and scaling-up approaches | |
| CN108359636B (en) | Induction method for improving directed differentiation of pluripotent stem cells into myocardial cells | |
| US20180362923A1 (en) | Method for separating and extracting huc-msc from wharton's jelly tissue of umbilical cord | |
| EP3388512A1 (en) | Method for separating and culturing mesenchymal stem cells from wharton's jelly tissue of umbilical cord | |
| RU2015131839A (en) | SUSPENDING AND CLUSTERING OF HUMAN PLURIPOTENT CELLS FOR THEIR DIFFERENTIATION IN PANCREATIC ENDOCRINE CELLS | |
| McGuckin et al. | Embryonic-like stem cells from umbilical cord blood and potential for neural modeling | |
| JP2012139246A5 (en) | ||
| EP1954803B1 (en) | Multipotent adult stem cells having an ability of oct4 expression derived from umbilical cord blood and method for preparing the same | |
| CN105969720B (en) | A kind of Human vascular endothelial's cell culture fluid and its cultural method | |
| RU2010103046A (en) | CULTIVATION OF SEPARATE PLURIPOTENT STEM CELLS | |
| CN103667349B (en) | Method for efficiently acquiring inductive pluripotent stem cells (iPSCs) | |
| US20200239842A1 (en) | Preparation and Expansion Methods for Human Pluripotent Stem Cell-Derived Human Retinal Pigment Epithelial Cells | |
| WO2013104909A4 (en) | Methods of preparing cells and compositions | |
| Liu et al. | Muse cells, a new type of pluripotent stem cell derived from human fibroblasts | |
| ES2703751T3 (en) | Formulation and method of passage and collection of pluripotent stem cells | |
| CN106520687A (en) | Method for differentiating induced pluripotent stem cells into mesenchymal stem cells | |
| CN103667184A (en) | In vitro culturing method of goat skeletal muscle satellite cells | |
| CN105385651B (en) | Inductive pluripotent stem cells Induction of committed differentiation is method and the liver cell of liver cell | |
| CN112553155A (en) | Umbilical cord blood mesenchymal stem cell culture method | |
| De Chiara et al. | Characterization of human mesenchymal stem cells isolated from the testis | |
| WO2007047581A3 (en) | Pulmonary stem cells, related methods and kits | |
| CN106381282A (en) | Induced pluripotent stem cell subculture method | |
| CN107523539A (en) | The induction of the lonely female class epiblast stem cell of mammal and culture method and obtained lonely female class epiblast stem cell line | |
| CN110804585A (en) | Separation method of umbilical cord mesenchymal stem cells | |
| CN106119191A (en) | A kind of placental villi plate mescenchymal stem cell and clinicization preparation method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |